Outline

Objective: Malignant gliomas are highly vascularized tumors. Therefore, antiangiogenic therapies emerge as a promising approach to improve the prognosis in these patients. The Ras-Raf system is activated by various growth factors and may enhance tumor and endothelial cell growth and motility. The aim of our study is to evaluate the effects of small inhibitory RNA (siRNA) directed against Raf-1 on glioma and endothelial cell viability, proliferation and motility.

Methods: Three different Raf-1 siRNA sequences and non-functional control siRNA were obtained from Dharmacon and Invitrogen. Human U373 glioma cells as well as human cerebral microvascular endothelial cells (HCMEC) were transfected with Raf-1 siRNA using a modified lipofectamine protocol. RT-PCR and Western blotting were used to control siRNA-mediated Raf-1 knock down on mRNA and protein level, respectively. Functional effects on viability and proliferation were evaluated in both cell types by the MTT assay and cell counting. The effects on cell specific motility were investigated by a spheroid based migration assay (U373) and a tube formation assay on Matrigel (HCMEC). For all assays, pure lipofectamine and non-functional siRNA served as controls.

Results: Half-quantitative RT-PCR and Western blotting revealed pronounced siRNA efficacy in U373 and HCMEC with a significant reduction of Raf-1 transcript and protein in both cell types. In U373 glioma cells, Raf-1 down regulation did not affect proliferation or glioma cell migration at various siRNA concentrations. In HCMEC, however, a highly significant decrease of cell proliferation and a significant inhibition of tube forming ability were achieved by Raf-1 siRNA as compared to non-functional siRNA or vehicle controls.

Conclusions: Inhibition of the Ras-Raf pathway by Raf-1 siRNA caused a significant reduction of endothelial cell growth and motility. If this can be confirmed in transformed endothelial cells derived from malignant gliomas, this strategy appears suitable for future anti-angiogenetic approaches in the treatment of malignant gliomas.